COMPOSITIONS FOR USE IN IDENTIFICATION OF STRAINS OF E. COLI O157:H7

• US 20110166040A1
• Filed: 05/28/2009
• Published: 07/07/2011
• Est. Priority Date: 09/05/1997
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Original Assignee: Ibis Biosciences Incorporated (Abbott Laboratories)
• Inventors: Hannis, James C., Hofstadler, Steven A., Sampath, Rangarajan, Li, Feng, Blyn, Lawrence B., Eshoo, Mark W., Ecker, David J.
• Application Number:
  US12/995,425
• Publication Number:
  US 20110166040A1
• Time in Patent Office: Days
• US Class Current: 506/12
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention relates generally to strain typing of Escherichia coli O157:H7, and provides methods, compositions and kits useful for this purpose when combined, for example, with molecular mass or base composition analysis.

Claims

1. A composition, comprising at least one purified oligonucleotide primer pair that comprises forward and reverse primers about 15 to 35 nucleobases in length, wherein said forward primer comprises at least 70% identity with a sequence selected from SEQ ID NOs:

1-10, and wherein said reverse primer comprises at least 70% identity with a sequence selected from SEQ ID NOs;

11-20.

2. The composition of claim 1, wherein said primer pair is configured to hybridize with conserved regions VNTR regions of E. coli O157:

H7.

3. The composition of claim 1, wherein said primer pair is selected from the group of primer pair sequences consisting of:

SEQ ID NOS;

1;

11, 2;

12, 3;

13, 4;

14, 5;

15, 6;

16, 7;

17, 8;

19, 9;

19, and 10;

20.

4. A kit comprising the composition of claim 1.

5. The composition of claim 1, wherein said forward and/or reverse primer further comprises a non-templated T residue on the 5′

-end.

6. The composition of claim 1, wherein said forward and/or reverse primer comprises at least one molecular mass modifying tag.

7. The composition of claim 1, wherein said forward and/or reverse primer comprises at least one modified nucleobase.

8. The composition of claim 7, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

9. The composition of claim 7, wherein said modified nucleobase is a mass modified nucleobase.

10. The composition of claim 7, wherein said mass modified nucleobase is 5-Iodo-C.

11. The composition of claim 7, wherein said modified nucleobase is a universal nucleobase.

12. The composition of claim 11, wherein said universal nucleobase is inosine.

13. A kit, comprising at least one purified oligonucleotide primer pair that comprises forward and reverse primers about 15 to 35 nucleobases in length, wherein said forward primer comprises at least 70% identity with a sequence selected from SEQ ID NOs:

1-10, and wherein said reverse primer comprises at least 70% identity with a sequence selected from SEQ ID NOs;

11-20.

14. A method of determining a presence of E. coli O157:

H7 in at least one sample, the method comprising;

(a) amplifying one or more segments of at least one nucleic acid from said sample using at least one purified oligonucleotide primer pair that comprises forward and reverse primers that are about 20 to 35 nucleobases in length, and wherein said forward primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOs;

1-10, and said reverse primer comprises at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOs;

11-20 to produce at least one amplification product; and

(b) detecting said amplification product, thereby determining said presence of said E. coli O157;

H7 in said sample.

15. The method of claim 14, wherein the strain of said E. coli O157:

H7 is determined.

16. The method of claim 14, wherein (a) comprises amplifying said one or more segments of said at least one nucleic acid from at least two samples obtained from different geographical locations to produce at least two amplification products, and (b) comprises detecting said amplification products, thereby tracking an epidemic spread of said E. coli O157:

H7.

17. The method of claim 14, wherein (b) comprises determining an amount of said E. coli O157:

H7 in said sample.

18. The method of claim 14, wherein (b) comprises detecting a molecular mass of said amplification product.

19. The method of claim 14, wherein (b) comprises determining a base composition of said amplification product, wherein said base composition identifies the number of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof in said amplification product, whereby said base composition indicates the presence of E. coli O157:

H7 in said sample or identifies the strain of said E. coli O157;

H7 in said sample.

20. The method of claim 19, comprising comparing said base composition of said amplification product to calculated or measured base compositions of amplification products of one or more known E. coli bacteria or strains of O157:

H7 E. coli present in a database with the proviso that sequencing of said amplification product is not used to indicate the presence of or to identify said E. coli O157;

H7, wherein a match between said determined base composition and said calculated or measured base composition in said database indicates the presence of or identifies said E. coli O157;

H7.

21. A method of identifying one or more strains of E. coli O157:

H7 in a sample, the method comprising;

(a) amplifying two or more segments of a nucleic acid from said one or more strains of E. coli O157;

H7 in said sample with two or more oligonucleotide primer pairs to obtain two or more amplification products;

(b) determining two or more molecular masses and/or base compositions of said two or more amplification products; and

(c) comparing said two or more molecular masses and/or said base compositions of said two or more amplification products with known molecular masses and/or known base compositions of amplification products of known strains of E. coli O157;

H7 produced with said two or more primer pairs to identify said one or more strains of E. coli O157;

H7 in said sample.

22. The method of claim 21, comprising identifying said one or more strains of E. coli O157:

H7 in said sample using three, four, five, six, seven, eight or more primer pairs.

23. The method of claim 21, wherein said one or more strains of E. coli O157:

H7 in said sample cannot be identified using a single primer pair of said two or more primer pairs.

24. The method of claim 21, comprising obtaining said two or more molecular masses of said two or more amplification products via mass spectrometry.

25. The method of claim 21, comprising calculating said two or more base compositions from said two or more molecular masses of said two or more amplification products.

26. The method of claim 21, wherein said two or more primer pairs comprise two or more purified oligonucleotide primer pairs that each comprise forward and reverse primers that are about 20 to 35 nucleobases in length, and wherein said forward primers comprise at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS:

1-10, and said reverse primers comprise at least 70% sequence identity with a sequence selected from the group consisting of SEQ ID NOS;

11-20 to obtain an amplification product.

27. The method of claim 21, wherein said primer pair is selected from the group of primer pair sequences consisting of:

SEQ ID NOS;

1;

11, 2;

12, 3;

13, 4;

14, 5;

15, 6;

16, 7;

17, 8;

19, 9;

19, and 10;

20.

28. The method of claim 21, wherein said determining said two or more molecular masses and/or base compositions is conducted without sequencing said two or more amplification products.

29. The method of claim 21, wherein said one or more strains of E. coli O157:

H7 in said sample cannot be identified using a single primer pair of said two or more primer pairs.

30. The method of claim 21, wherein said one or more strains of E. coli O157:

H7 in a sample are identified by comparing three or more molecular masses and/or base compositions of three or more amplification products with a database of known molecular masses and/or known base compositions of amplification products of known strains of E. coli O157;

H7 produced with said three or more primer pairs.

31. The method of claim 21, wherein members of said primer pairs hybridize to conserved regions of said nucleic acid that flank a variable region.

32. The method of claim 31, wherein said variable region varies between at least two of said strains of E. coli O157:

H7.

33. The method of claim 31, wherein said variable region uniquely varies between at least five of said strains of E. coli O157:

H7.

34. A system, comprising:

(a) a mass spectrometer configured to detect one or more molecular masses of amplicons produced using at least one purified oligonucleotide primer pair that comprises forward and reverse primers about 15 to 35 nucleobases in length, wherein said forward primer comprises at least 70% identity with a sequence selected from SEQ ID NOs;

1-10, and wherein said reverse primer comprises at least 70% identity with a sequence selected from SEQ ID NOs;

11-20; and

(b) a controller operably connected to said mass spectrometer, said controller configured to correlate said molecular masses of said amplicons with one or more strains of E. coli O157;

H7 identities.

35. The system of claim 34, wherein said primer pair is selected from the group of primer pair sequences consisting of:

SEQ ID NOS;

1;

11, 2;

12, 3;

13, 4;

14, 5;

15, 6;

16, 7;

17, 8;

19, 9;

19, and 10;

20.

36. The system of claim 34, wherein said controller is configured to determine base compositions of said amplicons from said molecular masses of said amplicons, which base compositions correspond to said one or more strains of E. coli O157:

H7 identities.

37. The system of claim 34, wherein said controller comprises or is operably connected to a database of known molecular masses and/or known base compositions of amplicons of known strains of E. coli O157:

H7 produced with the primer pair.

38. A composition comprising at least one purified oligonucleotide primer 15 to 35 nucleobases in length, wherein said oligonucleotide primer comprises at least 70% identity with a sequence selected from SEQ ID NOs:

1-20.